Human α-Defensin 51–9 and Human β-Defensin 2 Improve Metabolic Parameters and Gut Barrier Function in Mice Fed a Western-Style Diet

Obesity and metabolic comorbidities are associated with gut permeability. While high-fructose and Western-style diet (WSD) disrupt intestinal barrier function, oral administration of human α-defensin 5 (HD5) and β-defensin 2 (hBD2) is believed to improve intestinal integrity and metabolic disorders. Eighty-four male C57BL/6J mice were fed a WSD or a control diet (CD) ± fructose (F) for 18 weeks. In week 13, mice were randomly divided into three intervention groups, receiving defensin fragment HD51–9, full-length hBD2, or bovine serum albumin (BSA)-control for six weeks. Subsequently, parameters of hepatic steatosis, glucose metabolism, and gut barrier function were assessed. WSDF increased body weight and hepatic steatosis (p < 0.01) compared to CD-fed mice, whereas peptide intervention decreased liver fat (p < 0.05) and number of hepatic lipid droplets (p < 0.01) compared to BSA-control. In addition, both peptides attenuated glucose intolerance by reducing blood glucose curves in WSDF-fed mice. Evaluation of gut barrier function revealed that HD51–9 and hBD2 improve intestinal integrity by upregulating tight junction and mucin expression. Moreover, peptide treatment restored ileal host defense peptides (HDP) expression, likely by modulating the Wnt, Myd88, p38, and Jak/STAT pathways. These findings strongly suggest that α- and β-defensin treatment improve hepatic steatosis, glucose metabolism, and gut barrier function.


Introduction
Gut barrier impairment has been associated with a number of chronic diseases, including obesity and associated metabolic diseases, such as type 2 diabetes [1], nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) [2,3].Bacterial translocation, enabled by deteriorated gut barrier function, in human obesity is aggravated in patients with type 2 diabetes compared to their weight-matched, normoglycemic counterparts [4].Still, the mechanisms, by which gut barrier function is impaired and how this might affect obesity-related comorbidities are largely unclear [5].We and others have shown that high fructose intake, along with high-fat and high-sugar diet, the so called Western-style diet (WSD), increase permeability of intestinal barrier in rodents [6][7][8] and likely also in humans [9][10][11].Gut barrier impairments lead to increased translocation of bacterial endotoxins and probably even whole bacteria from the gut into the portal vein system and liver, promoting liver inflammation and liver steatosis [12][13][14].Low-grade inflammation, which is a direct consequence of gut barrier impairment, has been recognized as a relevant risk factor for development of metabolic diseases [3,15].While the relation between gut barrier impairment and metabolic diseases has become clear during recent years, attempts to restore gut barrier function are limited and mostly speculative.Both exogenous factors, including dietary factors such as fiber [16,17] and probiotics [18], as well as endogenous factors, such as mucus or defensins, are conceptual treatment options.Defensins (also termed cryptidins in mice) are an important component in the intestinal barrier's first line of defense [19,20].Defensins are small, cationic peptides with a characteristic folded β-structure, stabilized by three intramolecular disulfide bonds.Accordingly, they are classified into alpha-, beta-, and delta-defensins [20,21], as disulfide bonds are formed in α-defensins at position 1-6, 2-4, and 3-5, whereas β-defensins contain three disulfide bonds at 1-5, 2-4, and 3-6 [22,23].While human α-defensins 5 and 6 are mainly expressed by Paneth cells in the small intestine, β-defensins are produced at epithelial surfaces and in the colon [20], but also in other organs such as liver [24].HD5 and HD6 are the predominantly expressed α-defensins in the small intestine, which also exhibit high antimicrobial activity against pathogenic bacteria in the intestinal lumen [25].Upon proteolytic degradation in the reduced small intestinal environment, HD5 is cleaved to numerous fragments with biological activity, of which especially fragment HD5 1-9 , consisting of nine amino acids (ATCYCRTGR), showed good antimicrobial activity in vitro [26].
Human studies revealed that the onset of obesity is associated with decreased intestinal α-defensin mRNA expression [27].Moreover, reduced intestinal α-defensin expression disturbed the composition of intestinal microbiota and led to bacterial overgrowth in the gut, and decreased intestinal mucosal integrity [28][29][30].Consistently, patients with (de-) compensated liver cirrhosis showed decreased intestinal expression of human α-defensins 5 (HD5) and 6 (HD6) [31].In mice, oral administration of human HD5 improved diet-induced hepatic steatosis and dyslipidemia, and increased ileal expression of the murine analog DEFA5 [32][33][34].In further mouse studies, overexpression of full HD5 peptide increased ileal bacterial diversity and treatment with HD5 1-9 fragment enhanced the abundance of Akkermansia muciniphila [26,35,36].Further, it has been demonstrated that treatment of mice with a modified HD5 1-9 fragment, termed D3, protected against obesity by suppressing appetite and regulating gut microbiota [37].Therapeutic effects on barrier function were also shown for β-defensins in mice, as systemic administration of human β-defensin 2 (hBD2) reduced weight loss and disease activity index in three different colitis models in mice [38].hBD2 is a low molecular weight peptide consisting of 64 amino acids with positive net charges in its primary structure, providing antimicrobial activity by binding to negatively charged molecules on the surface of bacteria [39].In vitro, hBD-2 has been found to reduce proinflammatory cytokines, such as interleukin 1-β (IL1-β) and TNF-α (TNF) in LPS-treated human peripheral blood mononuclear cells (PBMCs) [38].Furthermore, oral administration of hBD-2 reduced both alcohol-induced liver injury and graft-versus-host disease, in numerous mouse strains, by improving barrier function and thereby modulating host-microbe interactions and immunoreactivity [40,41].
While high-fructose and Western-style diet has been associated with intestinal barrier dysfunctions, and in particular impaired expression of host defense peptides (HDPs), oral administration of full-length hBD2 and HD5 has been found to improve intestinal barrier integrity and obesity-related comorbidities, respectively.In the present study, we examined the effects of oral administration of HD5 peptide fragment HD5 1-9 and full peptide βdefensin 2 (hBD-2) on intestinal barrier function and metabolic diseases in male C57BL/6J mice fed a high-fructose or Western-style diet.

Results
2.1.Oral HD5 1-9 and hBD2 Administration Partially Improve WSD ± F Induced Deterioration in Body Weight, Mesenteric Fat Tissue, and Hepatic Steatosis Eighteen-week feeding of high-fructose solution reduced total food intake in both CD-(p < 0.001) and WSD-fed mice (p < 0.05) compared to their nonfructose-fed counterparts.However, despite decreased food intake, energy intake was increased in mice fed either CDF (p < 0.01) or WSDF (p < 0.001).Mice fed WSD also consumed more energy than the CD-fed control group (p < 0.05) (Figure 1A).While body weight gain in g was only increased in WSDF mice (p < 0.01) (Figure 1B), weight development during the 18-week feeding period increased significantly from week 9 onwards in WSD ± F-fed mice (p < 0.05) compared to mice receiving CD (Figure 1C).Moreover, weight development continued to increase up to week 18 for both WSD (p < 0.01) and WSDF (p < 0.0001) (Figure 1C).In addition, fat tissue measurements revealed that mesenteric fat in g and mesenteric fat-tobody weight (mesFat-BW) ratio were only significantly increased in WSDF-fed (p < 0.01), whereas both mesenteric fat weight (p = 0.094) and mesFat-BW ratio (p = 0.088) only tended to be increased in WSD-fed mice (Figure 1D,E).These results point toward the fact that highfat and high-sugar diet increased body weight especially by accumulation of mesenteric fat tissue, whereas high consumption of fructose had no effect on body weight and fat tissue (Figure 1B,D).

HD5 1-9 and hBD2 Treatment Improves Intestinal Barrier Function by Regulating Tight Junction and Mucin Expression
As obesity and metabolic diseases such as NAFLD have been associated with impaired intestinal barrier [43], we next analyzed parameters of gut barrier function.Histological evaluations of the ileum and colon revealed that neither diets nor HD5 1-9 or hBD2 treatment had any effect on intestinal morphology (Figure S3).A Lac/Man permeability assay revealed that 6 weeks of HD5 1-9 administration reduced the Lac/Man ratio in both WSD-(p < 0.01) and WSDF-fed mice (p < 0.05).Similar patterns were observed for hBD2 in WSD-fed (p < 0.05) but not in WSDF-fed mice (p = 0.6) (Figure S4).
In summary, our results imply that both HD51-9 and hBD2 enhance intestinal barrier function and integrity by modulating different gut barrier components.

HD51-9 and hBD2 Administration Restores High-Fructose-and WSD-Induced Impairments in Host Defense Peptide Secretion
High-fructose diets have been shown to compromise Paneth cell function [45], so we next examined small intestinal HDPs.Assessment of ileal immunohistochemically DEFA5 staining, which is the murine analogue to HD5, revealed that CD-fed mice expressed DEFA5 protein at the base of crypts in Paneth cell granules (Figure 5A), whereas DEFA5 Another component of the intestinal barrier function is the mucus layer.Measurements in the ileum revealed that neither mucin (Muc) 1 nor Muc2 were affected by WSD ± F, whereas 18-week feeding of CDF tended to reduced ileal Muc2 expression (p = 0.074) (Figure 4E,F).Moreover, measurements in the colon revealed a reduction in Muc1 by trend (p = 0.061), when mice were fed a WSDF (Figure S5).CDF-fed mice exhibited a similar tendency to reduced colonic Muc2 expression (p = 0.073) (Figure S5).We were able to show that administration of HD5 1-9 induced ileal Muc1 gene expression in WSDF-fed mice (p < 0.05) (Figure 4E), and ileal Muc2 mRNA expression in WSD-(p < 0.05) and WSDF-fed mice by trend (p = 0.098) (Figure 4F).We observed similar trajectories for hBD2-treated mice with an apparent upregulation of ileal Muc2 expression in WSD-(p = 0.07) and WSDF-fed mice (p = 0.08).In contrast, peptide treatment had no effect on Muc1 or Muc2 expression in the colon (Figure S5).
In summary, our results imply that both HD5 1-9 and hBD2 enhance intestinal barrier function and integrity by modulating different gut barrier components.

HD5 1-9 and hBD2 Administration Restores High-Fructose-and WSD-Induced Impairments in Host Defense Peptide Secretion
High-fructose diets have been shown to compromise Paneth cell function [45], so we next examined small intestinal HDPs.Assessment of ileal immunohistochemically DEFA5 staining, which is the murine analogue to HD5, revealed that CD-fed mice expressed DEFA5 protein at the base of crypts in Paneth cell granules (Figure 5A), whereas DEFA5 protein levels were almost absent in mice fed a CDF, WSD, or WSDF (p < 0.05) (Figure 5A).Interestingly, hBD2 but not HD5 1-9 administration increased DEFA5 protein signal in WSD-and WSDF-fed mice (p < 0.001) (Figure 5A).In contrast to protein abundance, PCR measurements revealed that neither CDF nor WSD ± F had any effect on ileal Defa5 mRNA expression.Despite the lack of dietary impact on mRNA expression, oral administration of HD5 1-9 induced Defa5 expression in WSD-fed mice (p < 0.0001), a pattern that was not recapitulated in hBD2-treated mice (p = 0.2) (Figure 5B).
The Wnt signaling pathway is an important regulator of intestinal HDP defense.We therefore hypothesized that peptide treatment modulates Wnt signaling activity.Quantification in the ileum found that none of the diets affected Wnt signaling pathway activity (Table 1).Still, we were able to show that HD5 1-9 but not hBD2 modulated the Wnt signaling pathway, by inducing Wnt3 (p < 0.01), Wnt5a (p < 0.05), and Wnt9a (p < 0.01) gene expression (Table 1).While Wnt signal transmitting receptor LRP6 was not affected, HD5 1-9 induced intracellular transcription factor Tcf1 (p < 0.01) (Table 1).Next, we analyzed Mmp7 gene expression, as the enzyme is required for proteolytic activation of antimicrobial peptides.However, PCR measurements revealed that neither diets nor peptide treatments had any effect on Mmp7 mRNA levels (Table 1).In conclusion, these data point toward the fact that peptide treatment ameliorated small intestinal gut barrier function by regulating HDP defense and that the Wnt signaling pathway might be involved in HD5 1-9 -mediated effects.

HD5 1-9 and hBD2 Modulate HDP Defense In Vitro through the Myd88, p38, and Jak/STAT Signaling Pathways
There is evidence that HDPs provide several immunomodulatory effects through the TLR4/Myd88, p38/MAPK, and Jak/STAT pathways [48][49][50].Therefore, we next used an ex vivo small intestinal organoid cell model to evaluate whether these signal pathways are involved in HD5 1-9 -and hBD2-mediated effects on host HDP defense.

HD51-9 and hBD2 Modulate HDP Defense In Vitro through the Myd88, p38, and Jak/STAT Signaling Pathways
There is evidence that HDPs provide several immunomodulatory effects through the TLR4/Myd88, p38/MAPK, and Jak/STAT pathways [48][49][50].Therefore, we next used an ex vivo small intestinal organoid cell model to evaluate whether these signal pathways are involved in HD51-9and hBD2-mediated effects on host HDP defense.

Discussion
In the present study, we demonstrate that administration of the defensin peptides, HD5 1-9 or hBD2, improve metabolic parameters in obese mice, such as weight gain, hepatic steatosis, and glucose metabolism.Moreover, our results strongly suggest that defensin treatments enhance intestinal barrier function, including induction of tight junction protein expression and small intestinal HDPs.Organoid cell culture experiments corroborated that HD5 1-9 -and hBD2-dependent effects on HDP expression were mediated through the Myd88, p38, and Jak/STAT signaling pathways.
The 18-week feeding of WSD ± F increased body weight, mesenteric fat, and hepatic steatosis in mice, whereas CDF did not affect these parameters.These results are in contrast to findings of some previous studies from our group showing that C57BL/6J mice exhibited higher liver weight and hepatic steatosis when fed a high-fructose diet for 12 weeks [2,8].Interestingly, we showed that only 6-week treatment with hBD2 tended to reduce mesenteric fat in g and liver weight, whereas both HD5 1-9 and hBD2 reduced mesenteric fat cell area and fat cell size, along with liver fat accumulation in WSDF-fed mice.Concordantly, Li et al. [37] demonstrated that a 10-week treatment with the modified HD5 1-9 fragment, termed D3, reduced body weight, especially epididymal fat weight in high-fat diet (HFD)-fed mice.Moreover, D3 reduced food intake in mice by activating uroguanylin (UGN) [37], an anorexic hormone secreted by small intestinal enterochromaffin cells, suppressing appetite via the UGN-transmembrane receptor guanylyl cyclase 2C (GUCY2C) axis [51].Additionally, a 10-week treatment of HFD-fed mice with full-length HD5 mitigated dyslipidemia, hypercholesterolemia, and circulating fatty acid levels [34].However, full-length HD5 failed to change body weight or epididymal white fat tissue mass [34].In addition, hBD2 has been found to exhibit liver protective effects, as 6-week treatment of mice fed an ETOH-containing diet improved alcohol-associated liver dis-ease (ALD), including reducing plasma alanine transaminase (ALT) activity and inducing hepatic expression of IL-17a and IL-22 [40].
In agreement with improved liver function, we were able to show that oral administration of either HD5 1-9 or hBD2 attenuated diet-induced glucose intolerance in mice fed a WSDF, resulting in a lowering of blood glucose curves and a reduction in the AUC.Since type 2 diabetes mellitus (T2DM) and decreased HDP expression in the upper small intestinal tract and saliva was found to be associated in human studies [52,53], we were interested to quantify the effects of HD5 1-9 or hBD2 on glucose metabolism.Although we did not observe any effects on fasting blood glucose levels, HD5 1-9 and hBD2 improved blood glucose curves at 30 min after glucose administration in CDF-and WSDF-fed mice.Comparable findings were also reported by Larsen et al. [34], demonstrating that full-length HD5 treatment in high-fat diet (HFD)-fed mice did not change fasting blood glucose levels, but attenuated blood glucose curves at 15 and 30 min after glucose challenge.Similarly, 10-week treatment of HFD-fed mice with the modified HD5 1-9 fragment, D3, improved glucose clearance rate comparable to those of normal chow-fed mice, indicating that the HD5 1-9 fragment, D3, may improve obesity-induced insulin resistance [37].Similarly, hBD2 was found to regulate diabetic wound healing in rats, as hBD2-loaded Poly-lacticco-glycolic acid (PLGA) nanoparticles accelerated healing, which was associated with decreased Mmp9 and TNFa expression [54].However, our results provide evidence for the first time that hBD2, comparable to HD5 1-9 , also modulates glucoregulatory capacity in WSDF-fed mice and thus could be an interesting therapeutic approach for diet-induced disturbances in glucose metabolism.
In the present study, we further demonstrated that defensin treatment improves intestinal barrier function by regulating ileal and colonic tight junction and ileal mucin expression.There is evidence that both HD5 1-9 and hBD2 modulate gut barrier function during inflammatory processes.Specifically, the HD5-enriched diet has been shown to improve colonic ocln and ZO-1 protein expression in ethanol-induced and dextran sulfate sodium (DSS)-induced colitis in mice [33].Consistently, Zeng et al. [55] revealed that administration of a Lactococcus lactis recombinant NZ9000SHD5 strain, which continuously produces mature HD5, to DSS-treated mice improved epithelial barrier integrity by reducing plasma FITC-dextran.These effects were associated with enhanced ocln and ZO-1 gene expression.While our data suggested no effects of HD5 1-9 on ileal or colonic ocln expression, we consistently found an induction of ileal ZO-1 gene expression in WSD ± F-fed mice treated with either HD5 1-9 or hBD2.Furthermore, we demonstrated that defensin-treatment decreased the Lac/Man ratio in WSD ± F-fed mice, indicating improved gut barrier function [43].Concordantly, Han et al. [56] provided evidence that porcine β-defensin (pBD)2 decreased plasma FITC-dextran levels and enhanced ZO-1, ZO-2, Muc1, and Muc2 mRNA and protein expression in the colon of DSS-treated mice [56].Similarly, hBD2 induced gene expression of ocln, cldn1, and ZO-1 and enhanced transepithelial electrical resistance (TEER) in Candida albicans-transfected Caco-2 cells [57].A further in vitro model, using human keratinocytes, implied that human β-defensins modulate gene expression of several claudins by activating Ras-related C3 botulinum toxin substrate 1, atypical protein kinase C, glycogen synthase kinase-3, and phosphatidylinositol 3 kinase [58].
Consistent with our findings that showed induced Muc1 and Muc2 gene expression in the ileum of WSD ± F-fed mice by peptide-treatment, 10 weeks of HD5 fragment D3 administration in HFD-fed mice enhanced colonic mucus thickness [37].Furthermore, there is evidence that Muc2 and defensins regulate each other.Thus, Muc2 −/− mice exhibited impaired colonic β-defensin 2 mRNA expression and peptide localization, as compared with wildtype littermates [59].Complementarily, hBD2 induced Muc2 gene expression in Caco-2 and HT-29 cells [60].Moreover, hBD2 modulates intestinal morphology, as 10-day hBD2 treatment of trinitrobenzene sulfonicacid (TNBS)-induced colitis in mice attenuated macroscopic and microscopic histology of mouse colons [38].Collectively, these data allow us to conclude that HD5 1-9 and hBD2 exhibit potential to attenuate intestinal barrier dysfunction.
Another important factor for intestinal barrier function are host defense peptides (HDP), which have been considered as a first line of defense in the intestinal tract [61].Our data showed that both high fructose intake and WSD diet impaired several small intestinal HDPs, which were restored by defensin treatment.Consistently, Hodin et al. [27] showed that individuals with obesity exhibit decreased HD5 and lysozyme levels.Similarly, feeding rats with a HFD for 2 weeks resulted in bile-acid toxic effects on Paneth cells, resulting in decreased ileal lysozyme protein levels and reduced Defa5 and 6 mRNA expression [62].While Larsen et al. [34] found no effects of a standard HFD or HD5 treatment on Defa5 gene expression in the jejunum or ileum of mice, the present study and our previous work indicate that the combination of high-fat and high-sugar diets alters ileal HDP expression [2,8], which is in line with a recent study showing how WSD promotes Paneth cell dysfunction [63].Importantly, HD5 1-9 and hBD2 administration partly rescued this phenotype by inducing α-defensin mRNA expression both in vitro and in vivo.However, our results demonstrated that the DEFA5 protein signal was only increased in WSD-and WSDF-fed mice receiving hBD2.These findings point toward the fact that hBD2 but not HD5 1-9 treatment enhanced HDP protein formation.
Furthermore, we provide evidence that HD5 1-9 -but not hBD2-mediated effects on HDP gene expression in mice were associated with increased activity in the Wnt signaling pathway, as HD5 1-9 treatment induced Wnt3, 5a, 9a, and Tcf1 expression.The Wnt signaling pathway affects the final maturation and biological function of Paneth cells [64], and aberrant regulation of the Wnt signaling pathway has been associated with various diseases [65,66].For example, Crohn's disease patients showed reduced HD5 and -6 levels, which were associated with decreased Tcf1, Tcf4, and LRP6 [19,67], along with reduced Wnt signaling molecules Wnt1, -3, and -3a [68].In contrast, hyperactivation of the Wnt signaling pathway, especially by mutation in the adenomatous polyposis coli (APC) gene, has been associated with colorectal carcinogenesis [69].However, there is evidence that overexpression of Defa5 reduced tumorigenesis in mice by directly binding phosphoinositide 3-kinases (PI3K) subunits [70].
By using an organoid cell-culture model, we demonstrated that HD5 1-9 and hBD2 induce HDPs through the Myd88 signaling pathway in vitro.Recent studies revealed that bacterial-mediated regulation of HDP expression occurs in a TLR/Myd88-dependent manner [71,72].Thus, microbiota-free, TLR-or Myd88-deficient mice exhibited decreased Defa5 mRNA expression, which was recovered by treating with Lactobacillus lactis or TLR agonists [73].Moreover, activation of TLR2 and -4 by LPS and peptidoglycan-stimulated α-defensin-2 promoter activation in Caco-2, T84, and SW480 cells [74].Consistent with our data, Funderburg et al. [49] demonstrated that hBD3 induces expression of costimulating molecules such as CD80, 86 and 40 on monocytes by interactions with TLR-1 and -2, resulting in enhanced Myd88 signaling.These results suggest that TLR signaling is not restricted to microbial signals but also can be activated by HDPs.
Furthermore, we demonstrated that HD5 1-9 and hBD2 effects were dependent on the MAPK/p38 signaling pathway.In addition to data showing that hBD2 and hBD3 levels are regulated by probiotics via p38 and NF K B activity [75], there is evidence that defensins also modulate different immunology processes through p38 signaling.In vitro data revealed that hBD1-4 induced the secretion of angiogenic factor ANG of human dermal fibroblasts by increasing phosphorylation of p38 and c-Jun N-terminal kinases (JNK) [50].Complementarily, hBD-2, -3, and -4, and LL-37 increased IL-18 mRNA expression in human keratinocytes, which has been associated with anti-inflammatory effects [76].Similarly, HD6 has been characterized to improve the outcome of colorectal cancer in vivo, whereby in vitro data indicated that HD6 overexpression reduced cell proliferation and serpine-1 expression, by suppressing nuclear translocation of p38 and JNK [77].
The Jak/STAT signaling pathway mediates immune regulatory processes, such as cytokine expression and inflammatory response [78].We demonstrated that defensinmediated effects on HDP gene expression were dependent on STAT3 and STAT5 signaling.Concordantly, it has been reported that hBD3 enhanced phosphorylation of Jak2 and STAT3 in human dermal fibroblasts, resulting in increased production of angiogenic growth factors, which has been associated with improved wound healing [48].Moreover, salidroside, a biological active component with anti-inflammatory properties, regulated Defa5 and -6 expression in intestinal epithelial 6 cells (IEC-6) from rats by regulating STAT3 activity [79].Similarly, the absence of STAT5 in transgenic mice has been found to reduce Paneth cells and to increase predisposition of mice for Clostridium difficile infection [63].Moreover, STAT5 −/− mice exhibited deregulations in intestinal epithelial stem cells markers and an impaired crypt regeneration after radiation-induced mucositis [80], suggesting that STAT5 could be also required for Paneth cell differentiation and regulation of intestinal HDPs.
A possible limitation in the present study is that we did not analyze intestinal stability of HD5 1-9 and hBD2 in mice.There is evidence that peptides generally display a low bioavailability due to proteolytic degradation [81].Thus, the reduced form of HD5 full peptide HD5 red was found to be degraded into different fragments such as HD5 1-9 by intestinal proteases [26].However, fragment formation has been associated with enhanced antimicrobial activity of HD5, wherein especially the resulting fragment HD5 1-9 exhibited comparable antimicrobial activity to the full HD5 peptide [26].Further, evidence was obtained for the human neutrophil α-defensin 4 (HNP4) fragment 1-11 that neither proteolysis nor reduction further degraded the fragment nor affected antimicrobial activity [82].A comparable mechanism was also shown for β-defensins.Wendler et al. [83] demonstrated that reduced β-defensin 1 (hBD1 red ) was proteolytically degraded into different fragments, which exhibited increased antimicrobial activity and inhibited growth of Bifidobacterium breve.Nevertheless, oral administration of HD5 has been shown to provide therapeutic effects, as treatment with modified HD5 1-9 fragment D3 regulated appetite in obese mice [37], and oral administration of HD5 improved glucose tolerance [34], gut barrier function, and microbiota composition in mice after radiation-induced injury [84].Similarly, intranasal treatment of hBD2 improved experimental asthma [85] and oral hBD2 administration ameliorated alcohol-associated liver disease in mice [40].Therefore, the results of the present study and previous work point toward the fact that the HD5 1-9 fragment and hBD2 full peptide provide pharmacokinetically effects in vivo.
In summary, the present study strongly suggests that both HD5 1-9 -and hBD2-treatment ameliorate metabolic parameters such as hepatic steatosis and glucose metabolism but also improve intestinal barrier function, especially tight junction protein expression and small intestinal HDP defense, in mice fed a low-fiber WSD enriched with fructose.Moreover, in vitro data indicated that peptide-mediated effects on HDP expression were dependent on the Myd88, p38, and Jak/STAT signaling pathways.Our results suggest that HDPs could be an interesting therapeutic approach for obesity and obesity-related comorbidities, but it is important to mention that production and extraction of whole HDP structures are difficult and expensive.The use of defensin fragments such as HD5 1-9 represents an opportunity to produce lower-cost bioactive compounds more easily.Since the present and other animal studies did not observe side effects of HD5 or hBD2 treatment, it is conceivable that HDPs could be a useful therapeutic tool for clinical practice.There are still studies necessary, evaluating whether defensin treatment has comparable effects on metabolic parameters in humans and which doses are required for pharmacokinetic effects.

Experimental Setup, Animals, and Diets
Experimental setup was carried out with 84 male C57BL/6J mice.Three 6-8-weekold mice per cage were cohoused in a specific pathogen-free (SPF) barrier facility with a controlled 12-h light/dark cycle, accredited by the Association for Assessment and Accreditation for Laboratory Animal Care International.The local Animal Care and Use Committee approved all experiments (Regional Council Stuttgart 362/20 EM).Mice were randomly divided into four dietary groups (n = 21 per diet), receiving either a compositionally defined control diet (CD, Ssniff ® CD88137, Soest, Germany) or a Western-style diet (WSD, Ssniff ® TD88137, Soest, Germany) for 18 weeks.Autoclaved tap water either with or without supplementation of D-(-) fructose 30% weight/weight (F, 5.09 KJ/g (1.22 kcal/g), >99.5% purity, Carl Roth, Karlsruhe, Germany) was offered ad libitum to mice.The detailed compositions of administered diets are shown in Table 2. Food and fluid intake together with body weight were assessed weekly throughout intervention.After 12 weeks, gut barrier tests together with an oral glucose tolerance test (oGTT) were performed, and animals were randomly divided into three intervention groups (n = 7 per intervention and diet).Following these measurements, mice were daily gavaged in the morning with either the recently discovered and biologically active HD5 1-9 (1.2 mg/kg), or full peptide hBD2 (1.2 mg/kg), or 0.01% BSA in PBS as control for six weeks.To determine gavage volume (1.2 mg/kg bw), mice were weighed three times per week.At week 20, intestinal barrier tests and oGTT were repeated.Afterward, mice were anesthetized and blood, liver, mesenteric fat tissue, and gut tissue specimens were collected and stored, as previously described [8].

Synthesis and Purification of Peptide
Fragment HD5 1-9 and Full Peptide hBD2 HD5 1-9 was chemically synthesized by EMC Microcollections GmbH (Tübingen, Germany) and purified by precipitation.The structure of the HD5 1-9 was confirmed by RP-HPLC-ESI-MS (purity ≥ 90%) and HD5 1-9 was dissolved in PBS.Recombinant hBD2 was expressed in E. coli, as described in the patent (WO2010/007166), and provided by Defensin Therapeutics.Proper folding and disulfide bridge topology was verified as previously described [38].Recombinant hBD2 (endotoxin levels <0.05 EU/mL) was kept in its natural tertiary structure (purity ≥ 96%) and was dissolved in PBS.

Measurement of Intestinal Permeability
In week 13 and week 20, mice were fasted for 6 h (h) before the intestinal permeability tests were performed by using the lactulose (Carl Roth, Karlsruhe, Germany) to mannitol (Sigma-Aldrich, St. Louis, MO, USA) ratio as measured in urine.The procedure was performed as previously described [86].

RNA-Extraction and Quantification of Gene Expression by Real-Time PCR in Mouse Tissue and Organoid Cells
Extraction of total RNA was performed by using peqGOLD TriFast system (PEQLAB, Erlangen, Germany) for mouse tissue or ExtractME Total RNA Kit (blirt S.A., Hilden, Germany) for organoid cells.For cDNA synthesis, a reverse transcription system kit with random primers was used and real-time (RT)-PCR was performed by using oligonucleotide primers listed in Supplemental Table S1, as previously described [8].Absolute gene expression was calculated by normalizing copy numbers to housekeeping gene β-actin and relative gene expression was quantified by using the ∆∆-Ct method.

Statistical Analysis
GraphPad Prism software 7.0 (GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analysis.The Kolmogorov-Smirnov test was performed to examine data for normal distribution.For nonparametric data, the Kruskal-Wallis test followed by Dunn's test or one-way-ANOVA and Dunnett s post-test were used.Depending on Gaussian distribution, an unpaired t-test or Mann-Whitney test was performed to evaluate differences between two groups.To evaluate effects of time and intervention, a two-way ANOVA with Tukey's multiple comparisons test was performed.p-values of p < 0.05 were considered as statistically significant and p-values ranging between > 0.05 and < 0.1 were considered to indicate a trend.

Patents
Aesculus Bio (AB) ApS holds a matter-of-substance patent related to HD5 1-9 for pharmaceutical use.Defensin Therapeutics (DT) ApS holds patents for pharmaceutical use of hBD2.

Figure 2 .
Figure 2. Oral administration of antimicrobial peptides reduced hepatic steatosis in mice fed WSDF.Liver weight in g (A) and liver-to-bodyweight (Liver-BW) ratio (B) after an 18-week feeding period are shown.Representative images of HE staining of hepatic tissue.Scale bar: 50 µm (C).Liver fat in % (D) and total number of hepatic lipid droplets (E) are shown.Data are presented as means +/− standard error of the mean (n = 6-7; graphically indicated by ° for one sample each).Statistical analysis was performed by one-way ANOVA with Dunnett´s post-test (A,B) or by the Kruskal-Wallis test with Dunn's test (C,E).Significant differences are indicated: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(*) p-values ranging between > 0.05 and < 0.1 were considered to indicate a

Figure 2 .
Figure 2. Oral administration of antimicrobial peptides reduced hepatic steatosis in mice fed WSDF.Liver weight in g (A) and liver-to-bodyweight (Liver-BW) ratio (B) after an 18-week feeding period are shown.Representative images of HE staining of hepatic tissue.Scale bar: 50 µm (C).Liver fat in % (D) and total number of hepatic lipid droplets (E) are shown.Data are presented as means +/− standard error of the mean (n = 6-7; graphically indicated by • for one sample each).Statistical analysis was performed by one-way ANOVA with Dunnett s post-test (A,B) or by the Kruskal-Wallis test with Dunn's test (C,E).Significant differences are indicated: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(*) p-values ranging between > 0.05 and < 0.1 were considered to indicate a trend.Abbreviations: CD, control diet; F, fructose; HD5, human α-defensin 5; hBD2, human β-defensin 2; Liver-BW ratio, Liver-to-body weight ratio; WSD, Western-style diet.

Figure 3 .
Figure 3. HD51-9 and hBD2 attenuated glucose tolerance in mice fed high-fructose diets.Fasting blood glucose in mg/dl (A) and changes in blood glucose from administration of glucose to the 15, 30, 45, and 60 min mark (B) after the 18-week feeding period are shown.Changes in blood glucose after BSA or HD51-9 or hBD2 treatment in mice fed a CD (C), CDF (D), WSD €, or WSDF (F) for 18 weeks.Area under the curve (AUC) in mg/dl x h after the 18-week feeding period (G) and after peptide treatment (H) are shown.Data are presented as means +/− standard error of the mean (n = 6-7; graphically indicated by ° for one sample each).Statistical analysis was performed by one-way ANOVA with Dunnett´s post-test (A) or by two-way ANOVA with Tukey's multiple comparisons

Figure 3 .
Figure 3. HD5 1-9 and hBD2 attenuated glucose tolerance in mice fed high-fructose diets.Fasting blood glucose in mg/dL (A) and changes in blood glucose from administration of glucose to the 15, 30, 45, and 60 min mark (B) after the 18-week feeding period are shown.Changes in blood glucose after BSA or HD5 1-9 or hBD2 treatment in mice fed a CD (C), CDF (D), WSD (E), or WSDF (F) for 18 weeks.Area under the curve (AUC) in mg/dL × h after the 18-week feeding period (G) and after peptide treatment (H) are shown.Data are presented as means +/− standard error of the mean (n = 6-7; graphically indicated by • for one sample each).Statistical analysis was performed by one-way ANOVA with Dunnett s post-test (A) or by two-way ANOVA with Tukey's multiple comparisons test (B-F).For nonparametric data, the Kruskal-Wallis test with Dunn's test was performed (G,H).Significant differences are indicated: * p-value < 0.05; ** p-value < 0.01; **** p-value < 0.0001.(*) p-values ranging between >0.05 and <0.1 were considered to indicate a trend.Abbreviations: AUC, area under the curve; CD, control diet; F, fructose; HD5, human α-defensin 5; hBD2, human β-defensin 2; WSD, Western-style diet.

Table 1 .
Regulators of antimicrobial peptides in ileal tissue.

Table 2 .
Nutrient formulation of experimental diets fed to male C57BL/6J mice for 18 weeks.